Project Summary/Abstract The broad goal of this proposal is to develop a general and highly-sensitive method that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases across the entire genomes of mammalian cells and Drosophila. Preliminary results from this Global nuclear Run-On and massively parallel Sequencing method (GRO-seq) demonstrate the potential of this approach to provide a global picture of the transcription status of genome. Additionally, these early results uncovered novel antisense short transcription units upstream and adjacent to many known promoters and suggest modes of regulation that heretofore have not been considered. This proposal seeks to rigorously test and further develop the GRO-seq method to provide unprecedented, near-nucleotide resolution of RNA polymerase positions over the genome. Aim 1 is to optimize and critically evaluate the first generation GRO-seq method, which is already producing results. Simple variations of this procedure should be capable of identifying the type of polymerase, that is, RNA polymerases I, II, or III. Aim 2 is to develop second generation, ultra-high resolution versions of GRO-seq that map with several-nucleotide resolution the 3'ends of nacent RNA, which are associated with active site of all transcriptionally- engaged polymerases. An additional adaptation of GRO-seq will identify transcription start sites of transcriptionally- engaged RNA polymerase II (Pol II) with high sensitivity. The precise position and status of Pol II relative to transcription start sites will identify sequences where transcription elongation is confronted with a rate limiting step. Indeed, recent studies are documenting that Pol II pausing immediately downstream of promoters is a rate-limiting step in the regulation of many metazoan genes. Furthermore, the correlation of Pol II position to genome-wide transcription factor maps will identify candidate factors involved in this regulation. Aim 3 will develop GRO-seq methods so they can be generally applied to key model systems including representative classes of human cell lines, mouse embryonic stem cells, and Drosophila embryos. This last aim will also test the ability to globally detect rapid changes in transcription in response to regulatory signals. Finally, although the focus of the proposal is to develop robust, generally-applicable assays of transcriptionally-engaged RNA polymerases, the data generated in testing these cell and organismal models will also provide critical transcription information to existing the human ENCODE and model organism modENCODE projects as well as to the mouse stem cell efforts of the International Regulome Consortium. Because GRO-seq can define engaged RNA polymerases in cells under normal conditions as well as during infection and disease, it provides a valuable tool for basic research of numerous diseases and can produce critical signatures in diagnostic studies. Project Narrative This is a proposal to develop a novel method that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases across the genome at unprecedented, near-nucleotide resolution. GRO-seq will provide a general tool that will be applicable to efforts of ENCODE and modENCODE projects to define in molecular terms how the DNA code in higher eukaryotes is read and regulated to provide the patterns of gene expression required to develop and maintain a multicellular organism. GRO-seq can define both the level and status of all engaged RNA polymerases in cells under normal conditions, and during infection and disease providing a powerful tool for basic and diagnostic studies.